Positional Cloning Of Nm4(Non-molting In The 4^st Instar) And Mini Gene In Silkwrom,Bombyx Mori

Bombyx mori is a good model organism for lepidopteran insects.It not only has high economic value,but also has important research significance.Now it has become the first research material for many biological researchers to study biological inheritance.Many researchers have achieved fruitful results in the study of the genetic characteristics of silkworm and the function of mutation control genes in various mutants of silkworm.Bombyx mori is a complete metamorphic insect.In the process of the metamorphic development of silkworm,each growth stage needs to undergo a molting,which is a very important growth process of silkworm in its whole life.The conditions that each silkworm must possesses from egg hatching to pupate.The new non-molting in the 4^thinstar?nm4?mutant and mini mutant silkworm are discovered from the silkworm variety S8V at the same time.The mutant develops normally in the 1^st,2^nd and 3^rd instar and molts on time,but it cannot molt and exuviate in the beginning of the pre-molting stage of the 4^th instar and finally dies.The mutant size of mini mutant is far less than the normal development stage silkworm.The size of mini mutant in 4^th instar is same with the 2^nd instar normal silkworm,The mini mutants can be distinguished by the 2^nd instar and are developed orderly but slow,and eventually die in the latter half of 4^th instar.In this study,the genetic analysis of nm4 and mini mutant was carried out to determine the mutant genes are inherited independently.Then the positional cloning of the mutant gene by map-based cloning was performed to construct a genetic linkage map.The main contents are as follows:1.Genetic analysis of nm4 mutantA silkworm moth isolated from nm4 mutant was selected for investigation in the S8V strain of Bombyx mori.The separation ratio between normal and nm4 was 3:1;The mutant develops normally in the 1st,2nd and 3rd instar and molts on time,but in the beginning of the pre-molting stage of the 4^th instar,the mutant larvae lasts for 6-8 days with cannot molt and exuviate and finally dies.Therefore,the mutant was subcultured using a hybrid.The reciprocal cross between hybrid?+nm4?and normal silkworm shows that the F₁offspring were normal.The F₂ offspring appeared the separation phenomenon.There were two phenotypes of normal silkworm and nm4.The separation ratio was 3:1;the backcross offspring between the normal parent type and F1 offspring were normal,while the backcross offspring between the hybrid?+nm4?of F₂ offspring and F₁ showed both phenotypes of normal and nm4 silkworm,the separation ratio was 3:1.In conclusion,we proved that the nm4 mutant was controlled by a single autosomal recessive gene.2.Positional cloning of nm4 mutant geneThe population of genetic analysis including parental?P1,P2?,first-filial generation?F₁?,Two types of backcross generation?BC₁F?BC₁M?were conducted.Two kinds of parental and hybrid populations were used for experiments to find 28 pairs of polymorphic SSR markers on the chromosomes of the silkworm.Then a chain verification experiment was performed on 10 pairs of normal and mutant type isolated from the BC₁F population.The results indicated that the nm4 gene was located on the chromosome 12 of silkworm.We used 340 nm4 mutants of BC₁M individuals to performed fine mapping.The results showed that the nm4 gene was located between two polymorphic SSR markers S2990-82 and S2990-157.The distance between the two markers was 0.40cM and the physical distance was about 320.3kb,12 candidate genes within the particular locus.The function of these 12 candidate genes was found by consulting the NCBI database.In addition,we replenished the mutant with ecdysone 20E lead to the mutant can developed molt to 5^th instar.This indicates that the mutant is defective in ecdysone level,so that the mutant can not develop normal molting.3.Genetic analysis of mini mutantThe new mini mutant and nm4 mutant silkworm is discovered from the silkworm variety S8V at the same time.The mutant develops normally in the 1^st instar,but gradually began to show difference from normal silkworms after the molting stage of the 1^st instar.The specific performance including the size of mini mutant is far less than the normal development stage silkworm,poor adaptation to the environment and the amount of food eaten was decreased.The same method of genetic analysis with nm4 revealed that mini mutant was controlled by a single autosomal recessive gene.4.Positional cloning of mini mutant geneThe same methods of genetic analysis with nm4 revealed that mini mutant was controlled by a single autosomal recessive gene of Bombyx mori,showing homozygous death.Two heterozygous genotypes were selfed and 3:1 segregation appeared in their offspring.However,there are fewer mini mutants in the offspring of hybrid with wild-type,which can not reach a ratio of 3:1.The unknown factors may affect the isolation of mutants in the cross-generation.The positional cloning results indicated that the mini gene was located on the chromosome 22 of Bombyx mori.The new nm4 mutant and mini mutant silkworm is discovered from the silkworm variety S8V at the same time and their common point was abnormal growth.The subject of this study identified them as new independent mutants,controlled by different genes.It is positive significance to further tap the function of the gene and use it as a target gene to control lepidopteran pest populations.